Cheng X, Sardana R, Kaplan H, Altosaar I
Agricultural Biotechnology Laboratories, Department of Biochemistry, Faculty of Medicine, University of Ottawa, 40 Marie Curie Private, Ottawa, Ontario, K1N 6N5 Canada.
Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):2767-72. doi: 10.1073/pnas.95.6.2767.
Over 2,600 transgenic rice plants in nine strains were regenerated from >500 independently selected hygromycin-resistant calli after Agrobacterium-mediated transformation. The plants were transformed with fully modified (plant codon optimized) versions of two synthetic cryIA(b) and cryIA(c) coding sequences from Bacillus thuringiensis as well as the hph and gus genes, coding for hygromycin phosphotransferase and beta-glucuronidase, respectively. These sequences were placed under control of the maize ubiquitin promoter, the CaMV35S promoter, and the Brassica Bp10 gene promoter to achieve high and tissue-specific expression of the lepidopteran-specific delta-endotoxins. The integration, expression, and inheritance of these genes were demonstrated in R0 and R1 generations by Southern, Northern, and Western analyses and by other techniques. Accumulation of high levels (up to 3% of soluble proteins) of CryIA(b) and CryIA(c) proteins was detected in R0 plants. Bioassays with R1 transgenic plants indicated that the transgenic plants were highly toxic to two major rice insect pests, striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas), with mortalities of 97-100% within 5 days after infestation, thus offering a potential for effective insect resistance in transgenic rice plants.
通过农杆菌介导的转化,从500多个独立筛选出的潮霉素抗性愈伤组织中再生出了9个品系的2600多株转基因水稻植株。这些植株用来自苏云金芽孢杆菌的两个合成cryIA(b)和cryIA(c)编码序列的完全修饰(植物密码子优化)版本以及分别编码潮霉素磷酸转移酶和β-葡萄糖醛酸酶的hph和gus基因进行转化。这些序列置于玉米泛素启动子、花椰菜花叶病毒35S启动子和甘蓝Bp10基因启动子的控制之下,以实现鳞翅目特异性δ-内毒素的高水平和组织特异性表达。通过Southern、Northern和Western分析以及其他技术在R0和R1代中证明了这些基因的整合、表达和遗传。在R0植株中检测到高水平(高达可溶性蛋白的3%)的CryIA(b)和CryIA(c)蛋白的积累。对R1代转基因植株的生物测定表明,转基因植株对两种主要的水稻害虫,即二化螟和三化螟具有高毒性,在接种后5天内死亡率为97%-100%,因此为转基因水稻植株提供了有效的抗虫潜力。